Sugar nucleotide regeneration beads (superbeads): a versatile tool for the practical synthesis of oligosaccharides.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
X Chen
Publications and source records attributed to X Chen.
Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma.
Patients suffering from nasopharyngeal carcinoma (NPC) generally exhibit elevated serum IgA antibody titres to Epstein-Barr virus (EBV) early antigen (EA) and virus capsid antigen (VCA). This property is frequently used as a diagnostic aid. Preliminary experiments suggested that an ELISA for IgA antibodies against the EBV-encoded thymidine kinase (TK) could form the basis of a more reliable diagnostic test. Here, we describe the construction of a recombinant baculovirus that expresses the EBV TK and present a full analysis of its use in serological surveys of NPC patients. Baculovirus-derived TK was used to develop a simple ELISA for serum IgA against this antigen. ELISA reactivity was strongly associated with NPC compared with an EBV-positive, normal control population. Comparison with the existing IgA-VCA and EA assays showed that the TK ELISA had higher sensitivity whilst the specificity was similar or higher. We conclude that the TK ELISA presents a strong predictor of NPC and, in its refined form, has improved pickup rates. In addition, results from patients with chronic nasopharyngitis (CNP) suggest that individuals with both symptoms of CNP and an elevated TK ELISA value may be at increased risk for the development of head-and-neck cancer.
Animals↗
Local structures of nanocrystalline GaN studied by XAFS.
X-ray absorption fine structure (XAFS) was used to investigate the local structures around Ga atoms in the hexagonal nanocrystalline and crystalline GaN under 78 and 300 K. For the first nearest neighbor coordination shell of Ga-N. the average bond length R (0.194 nm), coordination number N (4.0), thermal disorder sigma (0.0052 nm) and static disorder sigmaS (0.0007 nm) are neatly independent of the measured temperature and crystalline state. This indicates that the Ga-N covalent bond is much stronger, and the 4 nitrogen atoms in first nearest neighbor around Ga atoms keep the tetrahedral structure (Td). For the second nearest neighbor coordination shell of Ga-Ga, their bond lengths are about 0.318 nm. However, the sigmaS (0.0057 nm) of nanocrystalline GaN is 0.0047 nm larger than that of crystalline GaN (0.001 nm), and the sigmaT of nanocrystalline is 0.0053 nm and 0.0085 nm at the temperature of 78 and 300 K, respectively. The result indicates that the difference of local structure around Ga atoms between nanocrystalline and crystalline GaN occurs mainly at the Ga-Ga second nearest-neighbor coordination shell. The reason is explained as the local lattice distortion and unsaturated surface atoms existing in nanocrystalline GaN.
Journal Article↗
[STAT mediated thrombin induction of tissue inhibitor of metalloproteinase1 in human mesangial cells].
OBJECTIVE: To depict the role of signal transducers and activators of transcription (STAT) pathway in thrombin induction of tissue inhibitor of matrix metalloproteinase-1 (TIMP1) in human mesangial cells. METHODS: Human mesangial cells were isolated, seeded in RPMI1640, and stimulated with thrombin (0.5, 1.5, and 4.5 U/ml). Northern blot and electrophoretic mobility shift assay (EMSA) were employed to determine the levels of TIMP1 mRNA and STAT-DNA binding activity in the cells, respectively. In order to confirm the role of STAT in this cell event, STAT 1 and STAT 3 antisense oligonucleotides were employed to suppress their intra-cellular activity. RESULTS CONCLUSION: Cultured human mesangial cells expressed basal TIMP1 mRNA, and thrombin promoted TIMP1 gene transcription in dose-dependent manner. Hirudin, a specific inhibitor of thrombin, blocked thrombin-induced TIMP1 gene expression. Thrombin promoted STAT DNA binding activity in a manner parallel to TIMP1 mRNA expression, which was inhibited by its inhibitor, hirudin again. STAT1 and STAT3 antisense oligonucleotides inhibited both STAT DNA binding activity and TIMP1 mRNA expression in mesangial cells. CONCLUSION: STAT mediates the thrombin-induced expression of TIMP1 gene in cultured human mesangial cells.
Cells, Cultured↗
Stability and structure of RNA duplexes containing isoguanosine and isocytidine.
Isoguanosine (iG) and isocytidine (iC) differ from guanosine (G) and cytidine (C), respectively, in that the amino and carbonyl groups are transposed. The thermodynamic properties of a set of iG, iC containing RNA duplexes have been measured by UV optical melting. It is found that iG-iC replacements usually stabilize duplexes, and the stabilization per iG-iC pair is sequence-dependent. The sequence dependence can be fit to a nearest-neighbor model in which the stabilities of iG--iC pairs depend on the adjacent iG--iC or G--C pairs. For 5'-CG-3'/3'-GC-5' and 5'-GG-3'/3'-CC-5' nearest neighbors, the free energy differences upon iG-iC replacement are smaller than 0.2 kcal/mol at 37 degrees C, regardless of the number of replacements. For 5'-GC-3'/3'-CG-5', however, each iG--iC replacement adds 0.6 kcal/mol stabilizing free energy at 37 degrees C. Stacking propensities of iG and iC as unpaired nucleotides at the end of a duplex are similar to those of G and C. An NMR structure is reported for r(CiGCGiCG)(2) and found to belong to the A-form family. The structure has substantial deviations from standard A-form but is similar to published NMR and/or crystal structures for r(CGCGCG)(2) and 2'-O-methyl (CGCGCG)(2). These results provide benchmarks for theoretical calculations aimed at understanding the fundamental physical basis for the thermodynamic stabilities of nucleic acid duplexes.
Adenosine↗
p73 is transcriptionally regulated by DNA damage, p53, and p73.
p73 is a member of the p53 family. Recent studies have shown that DNA damage can stabilize p73 protein and enhance p73-mediated apoptosis in a c-Abl dependent manner. To determine what regulates p73 transcriptionally, we analysed the expression of p73 in several cell lines following genotoxic stresses. We found that p73 is induced in certain cell lines when treated with therapeutic DNA damaging agents. We also found that p53 and p73, but not mutant p53(R249S) and p73beta292, directly induce the expression of the p73 gene. In addition, we found one potential p53-binding site in the promoter of the p73 gene. This binding site is responsive to p53, p73, and DNA damage. Taken together, these data suggest that p73 is transcriptionally regulated by DNA damage and p53, and is autoregulated.
Antineoplastic Agents↗
Rapid kinetic and isotopic studies on dialkylglycine decarboxylase.
The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.
Alanine↗
Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy.
The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly defined to the well ordered state was monitored by time-resolved Fourier transform infrared spectroscopy (FTIR) for the first time. From the analysis of FTIR difference spectra, taken on time scales as short as 6 s and up to 1 h after addition of ethanol, intensity vs. time plots of an increasing band at 1618 cm(-1) were observed indicating formation of a beta-sheet coincident with the loss of intensity of a band at 1668 cm(-1) indicating decreases of random coil and/or silk I structure. Both infrared markers were fitted with identical biphasic exponential decay functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is indicated to either proceed sequentially through (at least) two intermediate states that contain different levels of beta-sheet structure or to have parallel pathways of initial beta-sheet formation followed by a slower 'perfection' phase. The first observed process forms in a burst phase a few seconds after mixing (or even faster), prior to the collection of the first spectrum at 6 s. The second observed process occurs with a time constant of approximately 0.5 min, the intermediate present at this stage then continues with a time constant of 5.5 min completing the observed formation of the beta-sheet. The conformation transition of this slower intermediate is not only indicated by an analysis of the kinetics of the random coil and beta-sheet-specific bands discussed above, it roughly coincides with the appearance of an additional infrared marker at 1695 cm(-1), which may be a marker for beta-sheet structure specific to the formation of the perfected structure. The conformation transition of this protein analyzed by infrared spectroscopy provides insight into a part of the fascinating process of cocoon formation in B. mori.
Animals↗
Aquaporin 3, a glycerol and water transporter, is regulated by p73 of the p53 family.
p73, a member of the p53 family, has been shown to exhibit similar biochemical activities to that of p53. However, in contrast to p53, p73 is rarely mutated in human tumors and p73 mutant mice develop neurological, pheromonal, and inflammatory defects, but not spontaneous tumors. Furthermore, p73 mutant mice are deficient in the physiological control of cerebral spinal fluid. To determine what mediates these p73 activities, cDNA subtraction assay was performed to identify cellular genes that are regulated by p73. We found that aquaporin 3 (AQP3), a glycerol and water transporter, is regulated by p73. In addition, we identified a potential p53 response element in the promoter of the AQP3 gene, which is responsive to p73. This suggests that AQP3 may mediate the activity of p73 in maintaining cerebral spinal fluid dynamics.
Aquaporin 3↗
[High frequency oscillatory ventilation with perfluorocarbon in rabbits with acute lung injury].
OBJECTIVE: To investigate the efficacy of high frequency oscillation with perfluorocarbon (PFC) in rabbits with acute lung injury (ALI). METHODS: Twenty four rabbits with ALI induced by repeated saline lung lavage were randomly assigned to four groups: conventional mechanical ventilation (CMV) group, CMV + PFC group, high frequency oscillation (HFO) group and HFO + PFC group. All rabbits were ventilated for two hours. Blood gases, respiratory system dynamic compliance (Cdyn) and intrapulmonary shunt fraction (Qs/Qt) were measured and calculated at 30, 60, and 120 min during experiment. RESULTS: Compared with the CMV, statistically improvements were shown in gas exchange, Cdyn and Qs/Qt at 30 min in the CMV + PFC group [PaO(2) 122 mm Hg +/- 15 mm Hg vs. 692 mm Hg +/- 82 mm Hg, PaCO(2) 29.1 mm Hg +/- 2.7 mm Hg vs. 47.1 mm Hg +/- 2.9 mm Hg, Cdyn (1.35 +/- 0.13) vs (0.97 +/- 0.13) ml.cm H(2)O(-1).kg(-1), Qs/Qt 18.1 +/- 1.1 vs.24.5 +/- 2.9] (P < 0.01 approximately 0.05) and remained stable for two hours. Compared with HFO, HFO + PFC improved oxygenation and ventilation more rapidly than HFO did. At 30 min intervals gas exchange and Qs/Qt were significantly improved (PaO(2) 174.9 +/- 18.5 vs 132.3 +/- 19.4, PaCO(2) 26.1 +/- 2.6 vs 34.9 +/- 4.6, Qs/Qt 17.1 +/- 2.4 vs 19.8 +/- 0.9) (P < 0.05)and remained stable to 60 min. The same differences were seen at 60 min. CONCLUSION: In rabbits with ALI, CMV + PFC could induce a significant improvement in gas exchange and Cdyn. HFO + PFC leads more rapidmy to reach best gas exchange and Cdyn.
Animals↗
Recalculation of the temperature inside capillaries using high buffer concentrations.
Explore the source record for details and available documents.
Buffers↗
Intestinal-enriched Krüppel-like factor (Krüppel-like factor 5) is a positive regulator of cellular proliferation.
Intestinal-enriched Krüppel-like factor (IKLF or KLF5) belongs to the family of mammalian Krüppel-like transcription factors. Previous studies indicate that expression of IKLF is enriched in the proliferating crypt epithelial cells of the intestinal tract. However, the biological function of IKLF is unknown. In the current study, we have shown that the level of IKLF mRNA was nearly undetectable in serum-deprived NIH3T3 fibroblasts but became acutely and significantly increased upon the addition of fetal bovine serum or the phorbol ester, PMA. This induction required protein synthesis because it was prevented by cycloheximide. Transfection of IKLF into NIH3T3 cells resulted in the formation of foci in a manner similar to that caused by the activated Ha-ras oncogene. Constitutive expression of IKLF in transfected NIH3T3 cells significantly increased the rate of proliferation when compared with cells transfected with an empty vector. The growth of IKLF-transfected cells was no longer inhibited by cell-cell contact or by low serum content. Moreover, these cells proliferated in an anchorage-independent fashion. We conclude that IKLF encodes a delayed early response gene product that positively regulates cellular proliferation and may give rise to a transformed phenotype when overexpressed.
3T3 Cells↗
5'-Deoxyadenosine contacts the substrate radical intermediate in the active site of ethanolamine ammonia-lyase: 2H and 13C electron nuclear double resonance studies.
The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of (2)H introduced from either deuterated substrate or deuterated coenzyme and of (13)C introduced into the ribosyl moiety of AdoCbl. A (2)H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of (2)H. Identical ENDOR frequencies were observed for (2)H irrespective of its mode of introduction into the complex. A (13)C doublet ENDOR signal was observed from samples prepared with [U-(13)C-ribosyl]-AdoCbl. The (13)C coupling tensor obtained from the ENDOR powder pattern shows that the (13)C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4+/-0.2 A between C1 of the radical and C5' of the labeled cofactor component. These results establish that the C5' carbon of the 5'-deoxyadenosyl radical moves approximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies approximately 12 A from the Co(2+) of cob(II)alamin. These findings are also consistent with the contention that 5'-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.
Binding Sites↗
Regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase by zinc.
The DPP1 gene, encoding diacylglycerol pyrophosphate (DGPP) phosphatase from Saccharomyces cerevisiae, has recently been identified as a zinc-regulated gene, and it contains a putative zinc-responsive element (UAS(ZRE)) in its promoter. In this work we examined the hypothesis that expression of DGPP phosphatase was regulated by zinc availability. The deprivation of zinc from the growth medium resulted in a time- and dose-dependent induction of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene. This regulation was dependent on the UAS(ZRE) in the DPP1 promoter and was mediated by the Zap1p transcriptional activator. Induction of the DGPP phosphatase protein and activity by zinc deprivation was demonstrated by immunoblot analysis and measurement of the dephosphorylation of DGPP. The regulation pattern of DGPP phosphatase in mutants defective in plasma membrane (Zrt1p and Zrt2p) and vacuolar membrane (Zrt3p) zinc transporters indicated that enzyme expression was sensitive to the cytoplasmic levels of zinc. DGPP phosphatase activity was inhibited by zinc by a mechanism that involved formation of DGPP-zinc complexes. Studies with well characterized subcellular fractions and by indirect immunofluorescence microscopy revealed that the DGPP phosphatase enzyme was localized to the vacuolar membrane.
Cell Membrane↗
The TRANSFAC system on gene expression regulation.
The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.
Binding Sites↗
The microsomal epoxide hydrolase Tyr113His polymorphism: association with risk of ovarian cancer.
Functional significance has been demonstrated in vitro for the exon 3 T-->C Tyr113His amino acid substitution polymorphism of the microsomal epoxide hydrolase (EPHX) gene. The higher activity or fast TT genotype was previously reported to be associated with an increased risk of ovarian cancer, and this association may reflect enhanced activation of endogenous or exogenous substrates to more reactive and mutagenic derivatives. Components of cigarette smoke are examples of exogenous substrates subject to such bioactivation, and smoking exposure may thus modify the risk associated with the EPHX polymorphism. We examined 545 cases of epithelial ovarian cancer and 287 unaffected controls for this EPHX T-C genetic variant to investigate whether, in the Australian population, the TT genotype was associated with (i) specific ovarian tumor characteristics; (ii) risk of ovarian cancer, overall or for specific subgroups; and (iii) risk of ovarian cancer in smokers specifically. Genotyping was carried out using the Perkin-Elmer ABI Prism 7700 Sequence Detection System for fluorogenic polymerase chain reaction allelic discrimination. Stratification of the ovarian cancer cases according to tumor behavior (low malignant potential or invasive), grade, stage, and p53 immunohistochemical status failed to show any heterogeneity with respect to the genotype defined by the EPHX polymorphism. There was a suggestion of heterogeneity with respect to histologic subtype (P=0.03), largely due to a decreased frequency of the TT genotype in endometrioid tumors. EPHX genotype distribution did not differ significantly between unaffected controls and ovarian cancer cases (overall, low malignant potential, or invasive) either overall or after stratification by smoking status. However, the TT genotype was associated with a decreased risk of invasive ovarian cancer of the endometrioid subtype specifically (age-adjusted odds ratio=0.38, 95% confidence interval=0.17-0.87). The results suggest that the proposed EPHX-mediated bioactivation of components of cigarette smoke to mutagenic forms is unlikely to be involved in the etiology of ovarian cancer in general but that a greater rate of EPHX-mediated detoxification may decrease the risk of endometrioid ovarian cancer. Mol. Carcinog. 30:71-78, 2001.
Adult↗
Composition and chirality of amino acids in aerosol/dust from laboratory and residential enclosures.
Initial results from the analyses of geological and anthropological samples for amino acids were difficult to accept because of the high enantiomeric purities of the analytes (i.e., predominantly L-amino acids). Consequently, sources of contamination had to be considered. All sources were eliminated except for direct atmospheric contamination. Essentially invisible, microscopic, aerosol/dust was found to rapidly contaminate the surface of samples and sample containers even after brief exposure times in clean laboratories. Contamination increased with exposure time. The aerosol/dust amino acids were contained predominantly in a proteinaceous material. Aerosol/dust from different locations can contain different percentages of proteinoid/amino acid material. However, the relative concentrations of the amino acids were similar for both laboratory and residential samples. The enantiomeric purity of the L-amino acids studied in aerosol/dust appears to be 99% or greater for the samples examined. Thus, even slight contamination of any sample with microscopic dust or aerosol particles can skew the results of trace amino acid analyses and amino acid e.e. determinations.
Aerosols↗
Quantitative determination of nitrendipine and its metabolite dehydronitrendipine in human plasma using liquid chromatography-tandem mass spectrometry.
A sensitive and high-throughput LC-MS-MS method was developed for simultaneous determination of nitrendipine (NIT) and its major metabolite, dehydronitrendipine (DNIT) in human plasma using nifedipine as the internal standard. Plasma samples were prepared based on a simple liquid-liquid extraction. The extracted samples were analyzed on a Zorbax SB C(18) column interfaced with a triple quadrupole tandem mass spectrometer. Positive atmospheric pressure chemical ionization was employed as the ionization source. The analytes were detected by use of selected reaction monitoring mode. Standard curves were linear (r > or = 0.995) over the concentration range of 0.4-40 ng/mL for NIT and 0.2-20 ng/mL for DNIT. The intra- and inter-run precision was measured to be below 8.5% for NIT and DNIT. The inter-run accuracy was less than 4% for the analytes. The overall extraction recoveries of NIT and DNIT were determined to be about 75% and 78% on average, respectively. The chromatographic run time was approximately 3 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of NIT and DNIT in a pharmacokinetic study of volunteers who received NIT orally.
Antihypertensive Agents↗